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Oral dietary developmental toxicity study with polyvinyl acetate phthalate (PVAP) in the rat
Regulatory Toxicology and Pharmacology 70 (2014) 325–332
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Oral dietary developmental toxicity study with polyvinyl acetate phthalate (PVAP) in the rat C.C. DeMerlis a,⇑, D.R. Schoneker a, J.F. Borzelleca b a b
Colorcon, Inc., 275 Ruth Road, Harleysville, PA 19438, USA School of Medicine, Virginia Commonwealth University, Richmond, VA 23229, USA
a r t i c l e
i n f o
Article history: Received 17 February 2014 Available online 30 July 2014 Keywords: PVAP PVAP-T Polyvinyl acetate phthalate Polyvinyl acetate phthalate and titanium dioxide Developmental study Enteric coating Enteric polymer Pharmaceutical excipient Modified release coating
a b s t r a c t Polyvinyl acetate phthalate (PVAP) was evaluated in a developmental toxicity study with Crl:CD(SD) rats. Female rats were provided continual access to the formulated diets on days 6 through 20 of presumed gestation (DGs 6 through 20) at concentrations of 0%, 0.75%, 1.5% and 3%. All surviving rats were sacrificed and Caesarean-sectioned on DG 21. The following parameters were evaluated: viability, clinical observations, body weights, feed consumption, necropsy observations, Caesarean-sectioning and litter observations, including gravid uterine weights, fetal body weights and sex, and fetal gross external, soft tissue and skeletal alterations. There were no treatment-related adverse effects reported in the developmental toxicity study. The maternal and developmental no-observable-adverse-effect level (NOAEL) of PVAP was the highest concentration administered, i.e., 3.0% (equivalent to 2324 mg PVAP/kg/day). Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Phthalate enteric polymers are used to form enteric coatings. These enteric coatings are used as delayed released tablet coatings that remain intact in the stomach then dissociate and release their contents in the small intestine. Their prime purpose is to delay the release of the active pharmaceutical ingredient which may be inactivated in the stomach or cause irritation of the gastric mucosa. Commonly used enteric polymers include polyvinyl acetate phthalate (PVAP). Polyvinyl acetate phthalate (PVAP) is a reaction product of phthalic anhydride, sodium acetate and partially hydrolyzed polyvinyl alcohol. It contains not less than 55% and not more than 62% of phthalyl (0-carboxybenzoyl C8H503) groups. The polyvinyl alcohol is a low molecular weight grade and is 87–89 mol percent hydrolyzed. Since the PVAP polymer is a partial esterification of partially hydrolyzed polyvinyl acetate, it may be represented as shown in Fig. 1. PVAP is also co-processed with titanium dioxide to produce polyvinyl acetate phthalate and titanium dioxide (PVAP-T). Polyvinyl acetate phthalate (PVAP) and co-processed polyvinyl acetate phthalate and titanium dioxide (PVAP-T) are enteric coating ⇑ Corresponding author. Fax: +1 215 661 2366. E-mail address: [email protected] (C.C. DeMerlis). http://dx.doi.org/10.1016/j.yrtph.2014.07.021 0273-2300/Ó 2014 Elsevier Inc. All rights reserved.
polymers used as pharmaceutical excipients and have been used in approved prescription and over-the-counter drug products in the United States and globally for many years. PVAP and PVAP-T are typically used in fully formulated dry powder coating systems manufactured by Colorcon, such as SuretericÒ and CoatericÒ, for rapid reconstitution providing an efficient method of applying enteric film coatings. Extensive toxicological testing of PVAP was conducted by Merck with PVAP in the 1960’s and Colorcon was able to secure reports of these studies. The reports did not describe the characterization, specifications or manufacturing process for the test material used in the studies. The studies were conducted consistent with generally accepted guidelines at that time. The studies were critically evaluated by experts and their findings published (Schoneker et al., 2003). A series of safety studies with PVAP manufactured by Colorcon to NF specifications which included a definitive 90-day sub-chronic toxicity study, a developmental toxicity study and several genotoxicity tests consistent with current Good Laboratory Practice (GLP) regulations and internationally recognized guidelines were conducted to provide additional safety data to support current and new applications for PVAP and PVAP-T. The results of the developmental toxicity study are reported herein. The 90-day sub-chronic toxicity study and the genotoxicity tests were accepted for publication in Food and Chemical Toxicology (DeMerlis et al., 2014).
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Fig. 1. PVAP chemical structure. Depending on the phthalyl content (a) will vary with (b) in mole percent. The acetyl content (c) remains constant depending on the starting material.
The purpose of this developmental toxicity study was to detect adverse effects of polyvinyl acetate phthalate (PVAP) on Crl:CD(SD) presumed-pregnant female rats and on embryo/fetal development consequent to exposure of the dam from implantation to closure of the hard palate. 2. Materials and methods This study was designed to evaluate ICH Harmonised Tripartite Guideline stages C and D of the reproductive process. The requirements of the U.S. Food and Drug Administration (FDA) and the Organisation for Economic Co-operation and Development were also used as the basis for study design (FDA Redbook, 2000; OECD 414, 2001; FDA, 2005; (ICH) S5(R2), 2005). 2.1. Test material Commercial samples of PVAP (NF) CAS No. 34481-48-6 were provided by Colorcon Inc. West Point, PA. The test material was a white to off-white powder and was stored at room temperature with a desiccant. Information documenting or certifying the identity, composition, strength, activity/purity and stability of the test article was provided by Colorcon to the testing facility. Neither the sponsor nor the study director was aware of any potential contaminants likely to have been present in the carrier that would have interfered with the results of this study. 2.2. Animals and husbandry Female rats Crl:CD(SD) were supplied from Charles River Laboratories, Inc., Portage, MI, USA. Male rats were used only for the purposes of breeding and are not considered part of the test system. The Crl:CD(SD) rat was selected as the test system because it is one mammalian species accepted and widely used world-wide for nonclinical studies of developmental toxicity (embryo-fetal toxicity/teratogenicity). This strain has been demonstrated to be sensitive to developmental toxicants and historical data and experience exist at the testing facility used for the study (Christian and Voytek, 1982; Christian, 1984; Lang, 1988). One hundred presumed pregnant Crl:CD(SD) rats with a mean age of 66 days were randomly assigned to four exposure groups (Groups I through IV), 25 rats per group. Mean weight (g ± SD) at study assignment (DG 0) was 231 ± 10.0. Upon arrival, rats were assigned to individual housing on the basis of computer-generated random units. Healthy, mated female rats were assigned to four exposure groups (Groups I through IV), 25 rats per group, using a computer-generated (weight-ordered) randomization procedure based on body weights recorded on DG 0. Male rats were given unique permanent identification numbers upon assignment to the testing facility’s breeder male rat population. Breeder rats were permanently identified using a tail tattoo.
Female rats were assigned temporary numbers at receipt and given unique permanent identification numbers when assigned to the study. Cage tags were marked with the study number, permanent rat number, sex, generation, test article identification, group number and dosage level. During the course of the study, individual rats were examined by the veterinary staff when needed. Medical treatments consisted of application of hydrogen peroxide to scabs. The study rooms were maintained under conditions of positive airflow relative to a hallway and independently supplied with a minimum of ten changes per hour of 100% fresh air that had been passed through 99.97% HEPA filters. Room temperature and humidity were monitored constantly throughout the study. Room temperature was targeted at 64 °F to 79 °F (18 °C to 26 °C); relative humidity was targeted at 30–70%. Rats were individually housed in stainless steel, wire-bottomed cages, except during the cohabitation period. During cohabitation, each pair of male and female rats was housed in the male rat’s cage. All cage sizes and housing conditions were in compliance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources). An automatically controlled 12-h dark:12-h fluorescent light cycle was maintained. Each dark period began at 1900 h (±30 min). Formulated diets of the test article, PVAP, in Certified Rodent DietÒ (meal form) #5002 (PMIÒ Nutrition International, St. Louis, MO, USA) were administered at a constant concentration of the test article in the diet (mg of PVAP/gram of diet). Female rats were given continual access to the formulated diets on days 6 through 20 of presumed gestation (DGs 6 through 20) at concentrations of 0%, 0.75%, 1.5% and 3%. All surviving rats were sacrificed and Caesarean-sectioned on DG 21. The following parameters were evaluated: viability, clinical observations, body weights, feed consumption, necropsy observations, Caesarean-sectioning and litter observations, including gravid uterine weights, fetal body weights and sex, and fetal gross external, soft tissue and skeletal alterations. 2.3. Test diet preparation Diets were prepared once at the testing facility, stored at controlled room temperature (20 °C to 25 °C) and used within the 43-day stability period. A constant concentration of the test article in the diet (mg of PVAP/gram of diet) was administered to the rats. Control group samples were determined to have no detectable level of PVAP. The formulations were within the acceptable limits of ±15% of theoretical concentrations and within the acceptable limits of ±5% RSD for homogeneity. Stability data for test article diet formulations bracketing the range of concentrations in this study were determined under the CRO’s protocol. Rats were provided ad libitum access to Certified Rodent DietÒ #5002 (PMIÒ Nutrition International, St. Louis, MO, USA) and/or prepared diets of Certified Rodent DietÒ #5002 and the test article in individual feeders. Dietary analyses revealed that no contaminants at levels exceeding the maximum concentration limits for certified feed or deviations from expected nutritional requirements were present. Local water that had been processed by passage through a reverse osmosis membrane (R.O. water) was available to the rats ad libitum from an automatic watering access system. Chlorine (sodium hypochlorite) was added to the processed water at a targeted concentration of 0.05 to 1.1 ppm as a bacteriostat. The processed water was analyzed twice annually for possible chemical contamination and monthly for possible bacterial contamination. Chewable NylabonesÒ were supplied to all rats during the course of the study. Environment enrichment is recommended by the Guide for the Care and Use of Laboratory Animals (1996). Analyses for possible contamination were conducted on each lot of NylabonesÒ and documented in the raw data.
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2.4. Experimental design Exposure Group
Concentrationa (%)
Number of rats
I II III IV
0.00 0.75 1.50 3.00
25 25 25 25
a The test article was considered 100% active/pure for the purpose of concentration calculations.
The oral (diet) route was selected for use because it is the proposed route for consumers. Dosages were selected on the basis of a 14-day range-finding toxicity study of PVAP administered in the diet to male and female Crl:CD SD rats. In that study, administration of PVAP in the diet for a minimum of 14 days was well tolerated in rats up to the highest level tested, 5% equivalent to 4.16 g/kg/day. Other concentrations tested were 0%, 1%, 2%, 3% PVAP. Mean test article consumption was 0.68, 1.40, 2.11, and 3.56 g/kg/day for treated males, and 0.80, 1.58, 2.44, and 4.16 g/kg/day for treated females. There was no test article-related mortality observed in this study. No test article-related clinical signs were noted. There were no toxicologically meaningful differences in body weight, body weight changes, food consumption, hematology parameters, coagulation parameters, clinical chemistry parameters, urinalysis parameters (macroscopic and microscopic), or absolute and relative organ weights in test article-treated males or females compared to controls. No test article-related gross findings were observed at scheduled sacrifice. Based on these results, the no-observedadverse-effect level (NOAEL) was determined to be 5% equivalent to 3.56 g/kg/day for males and 4.16 g/kg/day for females. Female rats were given continual access to the formulated diets on DGs 6 through 20. A constant concentration of the test article in the diet (mg of PVAP/gram of diet) was administered to the rats. The mg/kg/day dosages consumed were calculated based on the body weights and feed consumption values of the rats and presented for periods corresponding to body weight and feed consumption observations. 2.5. In-life observations After acclimation (5 days), 120 virgin female rats were cohabitated with 120 breeder male rats, one male rat per female rat. The cohabitation period consisted of a maximum of five days. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be DG 0 and assigned to individual housing. Mating was evaluated daily during the cohabitation period and confirmed by observation of spermatozoa in a smear of the vaginal contents and/or a copulatory plug observed in situ. Rats were observed for viability at least twice each day of the study and for clinical observations and general appearance weekly during the acclimation period and on DG 0. The rats were also examined for clinical observations, abortions, premature deliveries and deaths once daily during the exposure and postexposure periods. Body weights were recorded weekly during the acclimation period, on DG 0 and daily during the exposure and postexposure periods. Feed consumption values were recorded on DG 0 and daily during the exposure and postexposure periods. 2.6. Gross necropsy To minimize bias, Caesarean-sectioning, necropsy and subsequent fetal observations were conducted without knowledge of exposure group. Rats were euthanized by carbon dioxide asphyxiation. Live fetuses were euthanized by an intraperitoneal injection
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of sodium pentobarbital. Gross lesions were retained in neutral buffered 10% formalin for possible future evaluation. Unless specifically cited below, all other tissues were discarded. Representative photographs of maternal gross lesions and fetal gross, soft tissue and skeletal alterations are available in the raw data. All surviving female rats were euthanized on DG 21, Caesareansectioned and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. The gravid uterus was excised and weighed. One rat was apparently non-pregnant and the uterus was stained with 10% ammonium sulfide (Salewski, 1964) to confirm the absence of implantation sites. The uterus and ovaries of the apparently non-pregnant rat were retained in neutral buffered 10% formalin and were discarded when authorized by the study director. The number and distribution of corpora lutea were recorded. The uterus of each rat was excised and examined for evidence of pregnancy, number and distribution of implantation sites, early and late resorptions and live and dead fetuses. An early resorption was defined as one in which organogenesis was not grossly evident. A late resorption was defined as one in which the occurrence of organogenesis was grossly evident. A live fetus was defined as a term fetus that responded to stimuli. Non-responding term fetuses were considered to be dead. Dead fetuses and late resorptions were differentiated by the degree of autolysis present; marked to extreme autolysis indicated that the fetus was a late resorption. Placentae were examined for size, color and shape. Each fetus was removed from the uterus, placed in an individual container and individually identified with a tag noting the study number, litter number, uterine distribution and fixative. Each fetus was subsequently weighed and examined for sex and gross lesions. Approximately one-half of the fetuses in each litter were examined for soft tissue alterations, using a variation of the microdissection technique of Staples (Staples, 1974; Wilson, 1965). These fetuses were then fixed in Bouin’s solution and the heads were subsequently examined by free-hand sectioning; head sections were stored in alcohol. The decapitated carcasses were discarded. The remaining fetuses (approximately one-half of the fetuses in each litter) were eviscerated, cleared, stained with alizarin red S (Staples and Schnell, 1964) and examined for skeletal alterations. The fetuses were initially fixed in alcohol. Skeletal preparations were retained in glycerin with thymol added as a preservative. The rat that was sacrificed before scheduled termination due to adverse clinical observations was examined for the cause of condition on the day the observation was made. The rat was examined for gross lesions. The heart, lungs, liver, kidneys, stomach and spleen were retained in neutral buffered 10% formalin for possible histological evaluation. Pregnancy status and uterine contents were recorded. Conceptuses in utero were examined to the extent possible, using the same methods described for term fetuses. 2.7. Statistical analyses Data generated during the course of this study were recorded either by hand or using the Argus Automated Data Collection and Management System and the Vivarium Temperature and Relative Humidity Monitoring System. All data were tabulated, summarized and/or statistically analyzed. Averages and percentages were calculated. Litter values were used where appropriate. Clinical observations and other proportional data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution (Snedecor and Cochran, 1967a). Continuous data (e.g., body weights, body weight changes, feed consumption values, organ weights and litter averages for percent male fetuses, percent resorbed conceptuses, fetal body weights and fetal anomaly data) were analyzed using Bartlett’s Test of Homogeneity of Variances (Sokal and Rohlf, 1969a) and the Analysis of
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Variance (Snedecor and Cochran, 1967b), when appropriate [i.e., Bartlett’s Test was not significant (p > 0.001)]. If the Analysis of Variance was significant (p 6 0.05), Dunnett’s Test (Dunnett, 1955) was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett’s Test was significant (p 6 0.001)], the Kruskal–Wallis Test (Sokal and Rohlf, 1969b) was used, when less than or equal to 75% ties were present. In cases where the Kruskal–Wallis Test was statistically significant (p 6 0.05), Dunn’s Method of Multiple Comparisons (Dunn, 1964) was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher’s Exact Test (Siegel, 1956) was used to analyze the data. Count data were evaluated using the procedures described above for the Kruskal–Wallis Test (Sokal and Rohlf, 1969b). 3. Results There was no test article-related mortality. One female rat in the 0% exposure group was euthanized due to a broken hindlimb. All other rats survived to scheduled sacrifice. There were no test article related clinical or necropsy observations. Body weights, body weight gains, absolute and relative feed consumption and gravid uterine weights were unaffected by concentrations of PVAP in the diet as high as 3% (Figs. 2–4, Tables 1–3). The slight increase in feed consumption in the treated rats consuming PVAP may be due to the presence of the non-nutritive PVAP in the diet. No Caesarean-sectioning or litter parameters were affected by concentrations of PVAP in the diet as high as 3%. No gross external, soft tissue or skeletal fetal alterations (malformations or variations) were caused by concentrations of PVAP in the diet as high as 3%. Fetal ossification site averages were comparable among the four dosage groups and did not significantly differ.
450
Weight (g)
400 350 300
0.00% 0.75% 1.50% 3.00%
250 200
0
3
6
9 12 Days of gestation
15
18
Absolute consumaption (g)
Fig. 2. Maternal body weight.
35 30 25 20 0.00% 0.75% 1.50% 3.00%
15 10 5 0
0-6
6-9
9-12 12-15 Days of gestation
15-18
18-21
Fig. 3. Summary of maternal absolute feed consumption values.
100
Relative consumaption (g)
328
80 60 0.00% 0.75% 1.50% 3.00%
40 20 0
0-6
6-9
9-12
12-15
15-18
18-21
Days of gestation Fig. 4. Summary of maternal relative feed consumption values.
Consumed dosages averaged 567.0, 1139.1 and 2324.6 mg/kg/ day in Groups II, III and IV, respectively, for the entire dosage period (gestation days 6–20). 3.1. Mortality There was no test article-related mortality. One female rat in the 0% exposure group was euthanized due to a broken hindlimb. Observations in this rat are described in the following paragraph. All other rats survived to scheduled termination of the study. One rat in the 0% exposure group was euthanized due to adverse clinical observations on day 15 of gestation (DG 15). Adverse clinical observations occurred only on DG 15 and consisted of impaired righting reflex, decreased motor activity, chromodacryorrhea, chromorhinorrhea, lacrimation, limited use of both hindlimbs, limited gripping reflex in left hindlimb, moderate dehydration, cold to touch, tachypnea, urine-stained abdominal fur and scant feces. This rat lost 9.2% of the DG 13 body weight by DG 15. Feed consumption was reduced from DGs 13 to 15. Because these reduced values reflected this injury, body weights and feed consumption values on DG 14 and 15 were excluded from group averages and statistical evaluation. Necropsy revealed a fracture in the left fibula; all other tissues appeared normal. The litter consisted of eight embryos and six early resorptions. There was no obvious cause of the broken hindlimb; it is likely the rat got the limb caught in the cage and/or feed jar and injured itself trying to free it. 3.2. Clinical and necropsy observations
21
Adverse clinical observations were considered unrelated to PVAP because the incidences were not dosage dependent and/or the observations occurred in only one rat in a group. These clinical observations included sparse hair coat, chromorhinorrhea, scab on the nose, localized alopecia, bent tail, impaired righting reflex, decreased motor activity, chromodacryorrhea, lacrimation, limited use of hindlimb, limited gripping reflex of hindlimb, moderate dehydration, cold to touch, tachypnea, urine-stained abdominal fur and scant feces. Abnormal necropsy observations were considered unrelated to PVAP because the incidences were not dosage dependent and/or the observations occurred in only one rat in a group. The necropsy observations included large lymph nodes, numerous red areas on the thymus, slight dilation of the renal pelvis, large spleen and fractured fibula. Statistically significant increases (p 6 0.05 or p 6 0.01) in absolute and relative feed consumption in the 1.5% and 3% exposure groups on DGs 12 to 15, in relative feed consumption in the 1.5% and 3% exposure groups on DGs 9 to 12 and 6 to 21, and in relative feed consumption in the 3% exposure group on DGs 0 to 21 were
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C.C. DeMerlis et al. / Regulatory Toxicology and Pharmacology 70 (2014) 325–332 Table 1 Summary of maternal body weights and gravid uterine weights (tested rats/group: 25. pregnant rats/group: 25). Exposure group Concentrationa (%) Days of gestation
I 0.00 Maternal body weight (G)
II 0.75
III 1.50
IV 3.00
0 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 21Cd
231.5 ± 10.1 264.2 ± 11.5 269.0 ± 11.9 273.8 ± 11.7 279.7 ± 12.7 286.4 ± 12.8 292.6 ± 12.8 297.8 ± 12.5 304.4 ± 12.4 312.0 ± 12.5b,c 321.3 ± 12.6b,c 331.3 ± 12.2b,e 345.7 ± 12.8b,e 362.3 ± 12.8b,e 379.0 ± 13.4b,e 397.1 ± 13.7b,e 423.8 ± 15.7b,e 318.7 ± 13.5b,e
231.0 ± 10.2 263.8 ± 12.3 269.1 ± 13.5 273.3 ± 13.7 281.2 ± 14.2 287.9 ± 14.8 294.5 ± 15.7 301.1 ± 16.2 307.2 ± 16.0 313.4 ± 17.4 322.6 ± 18.3 333.1 ± 18.1 346.4 ± 19.9 363.0 ± 21.3 378.6 ± 22.2 395.9 ± 23.8 419.6 ± 23.1 317.0 ± 19.1
231.6 ± 9.5 265.4 ± 12.5 270.6 ± 13.2 276.4 ± 13.7 282.9 ± 14.1 289.1 ± 14.2 297.2 ± 15.0 302.6 ± 15.9 308.8 ± 16.2 314.9 ± 16.6 324.6 ± 17.4 335.6 ± 17.2 349.4 ± 18.7 365.6 ± 18.6 381.4 ± 19.8 399.1 ± 19.7 425.0 ± 21.8 320.4 ± 20.5
230.6 ± 11.0 263.3 ± 15.5 269.5 ± 15.4 274.9 ± 16.4 279.2 ± 17.2 285.6 ± 17.2 294.1 ± 18.2 300.0 ± 18.2 306.3 ± 19.0 313.2 ± 18.4 321.9 ± 19.7 332.4 ± 20.7 345.8 ± 20.9 362.0 ± 21.7 376.8 ± 23.2 392.4 ± 23.7 417.0 ± 24.8 317.1 ± 21.8
102.7 ± 13.3
104.6 ± 10.0
99.9 ± 18.2
Gravid uterine weight (G) 105.1 ± 9.9b,e a b c d e
Rats were given continual access to the formulated diets on days 6 through 20 of gestation. Average number of tested/pregnant rats: 24. Excludes values for dam 17553 that had reduced body weight values after 13 days of gestation reflecting an injury. Corrected maternal body weight (day 21 of gestation body weight minus the gravid uterine weight). Excludes values for dam 17553 that was sacrificed due to adverse clinical observations.
Table 2 Summary of maternal absolute feed consumption values (tested rats/group: 25; pregnant rats/group: 25 except 24 rats in group IV).
Table 3 Summary of maternal relative feed consumption values (tested rats/group: 25; pregnant rats/group: 25 except 24 rats in group IV).
Exposure group Concentrationa (%) Days of gestation
I II III 0.00 0.75 1.50 Maternal feed consumption (G/Day)
IV 3.00
Exposure group Concentrationa (%) Days of gestation
I II III 0.00 0.75 1.50 Maternal feed consumption (G/KG/Day)
IV 3.00
0–6 6–9 9–12 12–15 15–18 18–21 6–21 0–21
18.6 ± 1.5 20.9 ± 2.0 22.1 ± 1.9 22.6 ± 1.6b,c 24.9 ± 1.4b,d 24.8 ± 1.7b,d 23.1 ± 1.2b,d 21.8 ± 1.2b,d
18.4 ± 2.0 22.1 ± 2.7 23.7 ± 2.5 24.6 ± 2.8** 26.6 ± 3.5 24.9 ± 3.6 24.4 ± 2.4 22.6 ± 2.1
0–6 6–9 9–12 12–15 15–18 18–21 6–21 0–21
75.2 ± 4.9 76.8 ± 5.3 76.5 ± 4.9 73.3 ± 4.5b,c 73.2 ± 4.4b,d 63.5 ± 4.1b,d 71.7 ± 2.9b,d 68.9 ± 2.7b,d
74.5 ± 5.5 81.2 ± 7.3 81.6 ± 6.2** 79.0 ± 6.5** 77.9 ± 8.2 64.2 ± 8.0 75.8 ± 5.0** 71.7 ± 4.2**
18.7 ± 1.6 21.8 ± 2.1 23.0 ± 2.3 23.7 ± 2.2 25.7 ± 2.2 24.5 ± 2.4 23.7 ± 1.9 22.3 ± 1.7
18.7 ± 1.6 22.1 ± 1.5 23.4 ± 1.6 24.0 ± 1.8* 25.9 ± 2.4 25.2 ± 2.3b,e 24.0 ± 1.6b,e 22.4 ± 1.4b,e
75.4 ± 4.6 80.1 ± 6.1 79.1 ± 5.7 76.1 ± 5.0 75.4 ± 5.2 63.1 ± 5.7 73.7 ± 4.4 70.4 ± 3.6
75.3 ± 5.4 80.7 ± 4.9 79.7 ± 3.7* 76.8 ± 4.4* 75.4 ± 5.1 64.2 ± 4.5b,e 74.2 ± 3.2b,e,* 70.6 ± 2.5b,e
a Rats were given continual access to the formulated diets on days 6 through 20 of gestation. b Average number of tested/pregnant rats: 24. c Excludes values for dam 17553 that had reduced feed consumption values after day 13 of gestation reflecting an injury. d Excludes values for dam 17553 that was sacrificed due to adverse clinical observations. e Excludes values that were associated with spillage. * Significantly different from the Group I value (p 6 0.05). ** Significantly different from the Group I value (p 6 0.01).
a Rats were given continual access to the formulated diets on days 6 through 20 of gestation. b Average number of tested/pregnant rats: 24. c Excludes values for dam 17553 that had reduced feed consumption values after day 13 of gestation reflecting an injury. d Excludes values for dam 17553 that was sacrificed due to adverse clinical observations. e Excludes values that were associated with spillage. * Significantly different from the Group I value (p 6 0.05). ** Significantly different from the Group I value (p 6 0.01).
not considered to be toxicologically significant because decreased feed consumption, rather than increased, is the expected effect of a toxicant. There were 25 (100%), 25 (100%), 25 (100%) and 24 (96%) pregnant rats in Groups I through IV, respectively. As a result of the sacrifice of the control group dam with a broken hindlimb, Caesarean-sectioning observations were based on 24, 25, 25 and 24 pregnant rats in the four respective groups. There were no PVAP-related adverse effects on Caesareansectioning or litter parameters affected by concentrations of PVAP in the diet as high as 3% (Tables 4 and 5). The litter means for corpora lutea, implantations, preimplantation loss, litter sizes, live and dead fetuses, early and late resorptions, postimplantation loss, fetal body weights, percent dead or resorbed conceptuses, and percent
live male fetuses were comparable among the four dosage groups and did not differ significantly. There was one dead fetus in the 3% exposure group and one dam in the 3% exposure group that had a litter consisting of all late resorptions. These observations were not considered to be test article related because they were single events and not dose-dependent. One control group dam had three enlarged placentae (out of a total of 14). All other placentae appeared normal. Fetal alterations were defined as malformations (irreversible changes that occur at low incidences in this species and strain) or variations (common findings in this species and strain and reversible delays or accelerations in development). Litter averages were calculated for specific fetal ossification sites as part of the evaluation of the degree of fetal ossification.
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Table 4 Summary of Caesarean-sectioning observations. Exposure group Concentrationa (%)
I 0.00
II 0.75
III 1.50
IV 3.00
Rats tested Pregnant rats (number/%) Unscheduled sacrifice (number/%) Rats pregnant and caesarean-sectioned on day 21 of gestation
25 25/100 1/4 24
25 25/100 0/0 25
25 25/100 0/0 25
25 24/96 0/0 24
Corpora lutea
15.4 ± 1.6
14.9 ± 1.8
16.4 ± 2.8
14.7 ± 2.0
Implantations Preimplantation loss (%)
14.6 ± 1.4 4.9 ± 5.8
14.2 ± 1.9 4.4 ± 6.3
15.0 ± 1.4 6.7 ± 12.8
14.0 ± 1.6 4.1 ± 6.0
Litter sizes Live fetuses
14.1 ± 1.4 339 14.1 ± 1.4 0 0.0 ± 0.0
13.7 ± 2.1 343 13.7 ± 2.1 0 0.0 ± 0.0
14.1 ± 1.6 353 14.1 ± 1.6 0 0.0 ± 0.0
13.1 ± 3.2 314 13.1 ± 3.2 1 0.0 ± 0.2
Postimplantation loss (%)
0.4 ± 0.6 11 0.4 ± 0.6 0 0.0 ± 0.0 3.0 ± 4.4
0.5 ± 1.0 13 0.5 ± 1.0 0 0.0 ± 0.0 3.6 ± 6.8
0.9 ± 1.2 23 0.9 ± 1.2 0 0.0 ± 0.0 6.0 ± 7.5
0.9 ± 2.6 7 0.3 ± 0.6 15 0.6 ± 2.6 7.1 ± 20.4
Dams with any resorptions (number/%) Dams with all conceptuses dead or resorbed (number/%) Dams with viable fetuses (number/%) Placentae appeared normalb (number/%)
9/37.5 0/0.0 24/100.0 23/95.8
8/32.0 0/0.0 25/100.0 25/100.0
13/52.0 0/0.0 25/100.0 25/100.0
8/33.3 1/4.2 23/95.8 23/100.0
Dead fetuses Resorptions Early resorptions Late resorptions
% preimplantation loss = [(number of corpora lutea number of implantations)/number of corpora lutea] 100. % postimplantation loss = [(number of implantations number of live fetuses)/number of implantations] 100. a Rats were given continual access to the formulated diets on days 6 through 20 of gestation. b Excludes values for dams with all late resorptions.
Table 5 Summary of litter observations (Caesarean-delivered fetuses). Exposure group Concentrationa (%)
I 0.00
II 0.75
III 1.50
IV 3.00
Litters with one or more live fetuses
24
25
25
23
Implantations Live fetuses
14.6 ± 1.4 339 14.1 ± 1.4 52.6 ± 10.5 5.46 ± 0.23 5.58 ± 0.22 5.31 ± 0.25 3.00 ± 4.40
14.2 ± 1.9 343 13.7 ± 2.1 54.2 ± 14.2 5.53 ± 0.28 5.67 ± 0.30 5.37 ± 0.30 3.60 ± 6.80
15.0 ± 1.4 353 14.1 ± 1.6 49.8 ± 12.4 5.46 ± 0.25 5.62 ± 0.24 5.30 ± 0.25 6.00 ± 7.50
14.1 ± 1.6 314 13.6 ± 1.7 49.0 ± 14.3 5.50 ± 0.33 5.66 ± 0.36 5.35 ± 0.32 3.10 ± 5.30
Live male fetuses/litter (%) Live fetal body weights/litter (g) Males fetuses (g) Female fetuses (g) Dead or resorbed conceptuses/litter (%) a
Rats were given continual access to the formulated diets on days 6 through 20 of gestation.
Table 6 Summary of fetal observations. Exposure group Concentration (%)
I 0
II 0.75
III 1.5
IV 3
Litters with fetuses with alterations Numbers of fetuses with alterations Percent of fetuses with alterations Bifid centrum in thoracic vertebra (fetuses, litters)
6
5
6
3
7
7
6
3
2
2.2
1.7
0.9
2.2
1.1
4.4
1.1
Innominate artery absent
Innominate artery absent
Constricted tail; no anal opening
Moderate dilation of ureters
Innominate artery absent Short ribs
Interfrontal ossification site Cervical rib at 7th cervical vertebra
Folded retina Arches of 6th cervical vertebra had the appearance of the 7th
Incomplete ossified cervical arch Cervical rib at 7th cervical vertebra
Additional Observations
2 littermates with wavy ribs; one with incomplete ossified rib
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Fetal evaluations were based on 339, 343, 353 and 314 live, DG 21 caesarean-delivered fetuses in 24, 25, 25 and 23 litters in the 0, 0.75%, 1.5% and 3% exposure groups, respectively. Each of these fetuses was examined for gross external alterations. Of these respective fetuses, 161, 164, 170 and 150 fetuses were examined for soft tissue alterations, and 178, 179, 183 and 164 fetuses were examined for skeletal alterations and fetal ossification site averages. It was also possible to examine one dead fetus in the 3% exposure group. This specimen had a medially rotated left hindlimb and a laterally rotated right hindlimb at external examination and all tissues appeared normal at soft tissue examination. Values for this specimen were excluded from summarization and statistical analyses. There were no PVAP-related gross external, soft tissue or skeletal fetal alterations (malformations or variations). Mean fetal ossification sites averages were comparable among the four dosage groups and did not differ significantly. There were no statistically significant or biologically important differences among the four dosage groups in the average numbers of ossification sites per fetus for the hyoid, vertebrae (cervical, thoracic, lumbar, sacral and caudal), ribs, sternum (manubrium, sternal centers and xiphoid), forelimbs (carpals, metacarpals and phalanges) or hindlimbs (tarsals, metatarsals and phalanges). All values were within the ranges observed historically at the testing facility. Fetal observations are included in Table 6.
4. Discussion and conclusion PVAP, polyvinyl acetate phthalate, a phthalate enteric polymer, is a component of enteric coatings used in delayed release tablet coatings. PVAP is not a phthalate as defined by regulatory agencies such as the FDA and EPA. These agencies have defined phthalates as diesters of low molecular weight alcohols and orthophthalic acid (‘‘phthalic acid’’). ‘‘By contrast, phthalate enteric polymers are monoesters of orthophthalic acid and the alcohol is present in the form of a polymer backbone. These differences in structure translate to differences in chemical properties and biological activities.’’ ‘‘The common term in the nomenclature of these high molecular enteric polymers is the word ‘‘phthalate’’ because these enteric polymers have been modified by esterification with orthophthalic acid groups. The enteric polymers are large molecules with a typical number average (Mn) molecular weight in the range of 60,000– 130,000 Da.’’ By contrast, ‘‘esters of orthophthalic acid with low molecular weight alcohols, such as dibutyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) have been developed and used commercially as plasticizers. DBP and DEHP are small molecules with molecular weight of only 278 and 390, respectively’’ (Kossor et al., 2014). The large molecular weight phthalate enteric polymers are not bioavailable following oral administration (gavage, dietary). There are no data to support the metabolism (biotransformation) of these phthalate enteric polymers to bioactive metabolites. Data presented in published in vivo and in vitro studies with Cellulose Acetate Phthalate (CAP), CAP Aqueous Dispersion, Hypromellose Phthalate (HPMCP), and PVAP demonstrate the lack of biological activity of these phthalate enteric polymers. The inappropriate use of the term ‘‘phthalates’’ by some scientists and the media has created unwarranted safety concerns. It is noted, however, that when the FDA recommended to the pharmaceutical industry that it should avoid the use of phthalates, it referred to only two specific phthalates, DBP and DEHP. The weight of the available published evidence on phthalate enteric polymers supports the safety of their use in pharmaceutical preparations. The study reported herein adds further evidence of the biological inertness of these polymers. The toxicity of phthalate enteric
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polymers will not be compared to the toxicity of the phthalates used as plasticizers (e.g. DBP and DEHP) since these phthalates are neither chemically nor functionally equivalent to phthalate enteric polymers and such discussion would only further confuse the scientific literature and the public media. The purpose of this study, a classical developmental study, was to assess the potential developmental toxicity of PVAP in Crl:CD(SD) presumed-pregnant female rats (from implantation to closure of the hard palate). There were no deaths and no evidence of consistent, biologically and/or statistically significant, dosedependent adverse biological effects on any of the parameters evaluated in the dams or fetuses. The maternal and developmental no-observable-adverse-effect level (NOAEL) of PVAP is the highest concentration tested, i.e., 3.0% (equivalent to 2324.6 mg PVAP/kg/day). Conflict of interest C.C. DeMerlis and D.R. Schoneker are employees of Colorcon, Inc. Colorcon retained J.F. Borzelleca as a consultant and compensated him for his services. Acknowledgment The authors thank Hua Deng of Colorcon, Inc. for assistance in the preparation of the manuscript. References Christian, M.S., 1984. Reproductive toxicity and teratology evaluations of naltrexone. J. Clin. Psychiatry 45 (9 Sec 2), 7–10. Christian, M.S., Voytek, P.E., 1982. In Vivo Reproductive and Mutagenicity Tests. A Guide to General Toxicology. S. Karger, Basel (CH), pp. 295–325. DeMerlis, C.C., Schoneker, D.R., Borzelleca, J.F., 2014. Safety of PVAP and PVAP-T including a 90-day dietary toxicity study in rats and genotoxicity tests with polyvinyl acetate phthalate (PVAP). Food Chem. Toxicol. 70, 231–240. Dunn, O.J., 1964. Multiple comparisons using rank sums. Technometrics 6 (3), 241– 252. Dunnett, C.W., 1955. A multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 50, 1096–1121. Guidance for Industry: Nonclinical Studies for the Safety Evaluation of Pharmaceutical Excipients. U.S. Dept of Health and Human Services Food and Drug Administration (CDER) (CBER), Rockville (MD), 2005. Guideline for Industry: Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility (ICH) S5(R2), last update November, 2005. U.S. Dept of Health and Human Services Food and Drug Administration, Rockville (MD). Institute of Laboratory Animal Resources Commission on Life Sciences and the National Research Council, 1996. Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington (DC). Kossor, D., DeMerlis, C., Obara, S., Hernandez, A., Moreton, C., 2014. Eye on Excipients: Misunderstandings about Excipients that Contain ‘‘Phthalate’’ in Their Name. Tablets & Capsules. pp. 39–45. Lang, P.L., 1988. Embryo and Fetal Developmental Toxicity (Teratology) Control Data in the Charles River Crl:CDÒBR Rat. (Database provided by Argus Research Laboratories, Inc.). Charles River Laboratories, Inc, Wilmington (MA). OECD guidelines for the testing of chemicals. Prenatal developmental toxicity study, No. 414 section 4 Health Effects (Pink pages); last updated January 22, 2001. Organisation for Economic Co-operation and Development. Redbook, 2000. Toxicological Principles for the Safety Assessment of Food Ingredients: IV.C.9.b. Guidelines for Developmental Toxicity Studies; July, 2000. U.S. Dept of Health and Human Services Food and Drug Administration (CFSAN) (OFAS), Springfield (VA). Salewski, E.,1964. Färbemethode zum makroskopischen nachweis von implantations stellen am uterus der ratte. G [Staining method for macroscopic demonstration of implantation sites in the rat uterus]. Arch. Pathol. Exp. Pharmacol. 247, 367. Schoneker, D.R., DeMerlis, C.C., Borzelleca, J.F., 2003. Evaluation of the toxicity of polyvinyl acetate in experimental animals. Food Chem. Toxicol. 41, 405. Siegel, S., 1956. The Fisher’s Exact Probability Test. Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill Co, New York (NY), pp. 96–105. Snedecor, G.W., Cochran, W.G., 1967a. Variance Test for Homogeneity of the Binomial Distribution. Statistical methods, sixth ed. Iowa State University Press, Ames, pp. 240–241. Snedecor, G.W., Cochran, W.G., 1967b. Analysis of Variance. Statistical methods, sixth ed. Iowa State University Press, Ames, pp. 258–298.
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Sokal, R.R., Rohlf, F.J., 1969a. Bartlett’s Test of Homogeneity of Variances Biometry: The Principles and Practice of Statistics in Biological Research. Freeman & Co, San Francisco (CA), pp. 370–371. Sokal, R.R., Rohlf, F.J., 1969b. Kruskal-Wallis Test. Biometry: The Principles and Practice of Statistics in Biological Research. Freeman & Co, San Francisco (CA), pp. 388–391. Staples, R.E., 1974. Detection of visceral alterations in mammalian fetuses. Teratology 9 (3), A37–A38.
Staples, R.E., Schnell, V.L., 1964. Refinements in rapid clearing technic in the KOHalizarin red S method for fetal bone. Stain Technol. 39, 61–63. Wilson, J.G., 1965. Methods for administering agents and detecting malformations in experimental animals. In: Wilson, J.G., Warkany, J. (Eds.), Teratology: Principles and Techniques. University of Chicago Press, Chicago (IL), pp. 262– 277.
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Regulatory Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/yrtph
Oral dietary developmental toxicity study with polyvinyl acetate phthalate (PVAP) in the rat C.C. DeMerlis a,⇑, D.R. Schoneker a, J.F. Borzelleca b a b
Colorcon, Inc., 275 Ruth Road, Harleysville, PA 19438, USA School of Medicine, Virginia Commonwealth University, Richmond, VA 23229, USA
a r t i c l e
i n f o
Article history: Received 17 February 2014 Available online 30 July 2014 Keywords: PVAP PVAP-T Polyvinyl acetate phthalate Polyvinyl acetate phthalate and titanium dioxide Developmental study Enteric coating Enteric polymer Pharmaceutical excipient Modified release coating
a b s t r a c t Polyvinyl acetate phthalate (PVAP) was evaluated in a developmental toxicity study with Crl:CD(SD) rats. Female rats were provided continual access to the formulated diets on days 6 through 20 of presumed gestation (DGs 6 through 20) at concentrations of 0%, 0.75%, 1.5% and 3%. All surviving rats were sacrificed and Caesarean-sectioned on DG 21. The following parameters were evaluated: viability, clinical observations, body weights, feed consumption, necropsy observations, Caesarean-sectioning and litter observations, including gravid uterine weights, fetal body weights and sex, and fetal gross external, soft tissue and skeletal alterations. There were no treatment-related adverse effects reported in the developmental toxicity study. The maternal and developmental no-observable-adverse-effect level (NOAEL) of PVAP was the highest concentration administered, i.e., 3.0% (equivalent to 2324 mg PVAP/kg/day). Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Phthalate enteric polymers are used to form enteric coatings. These enteric coatings are used as delayed released tablet coatings that remain intact in the stomach then dissociate and release their contents in the small intestine. Their prime purpose is to delay the release of the active pharmaceutical ingredient which may be inactivated in the stomach or cause irritation of the gastric mucosa. Commonly used enteric polymers include polyvinyl acetate phthalate (PVAP). Polyvinyl acetate phthalate (PVAP) is a reaction product of phthalic anhydride, sodium acetate and partially hydrolyzed polyvinyl alcohol. It contains not less than 55% and not more than 62% of phthalyl (0-carboxybenzoyl C8H503) groups. The polyvinyl alcohol is a low molecular weight grade and is 87–89 mol percent hydrolyzed. Since the PVAP polymer is a partial esterification of partially hydrolyzed polyvinyl acetate, it may be represented as shown in Fig. 1. PVAP is also co-processed with titanium dioxide to produce polyvinyl acetate phthalate and titanium dioxide (PVAP-T). Polyvinyl acetate phthalate (PVAP) and co-processed polyvinyl acetate phthalate and titanium dioxide (PVAP-T) are enteric coating ⇑ Corresponding author. Fax: +1 215 661 2366. E-mail address: [email protected] (C.C. DeMerlis). http://dx.doi.org/10.1016/j.yrtph.2014.07.021 0273-2300/Ó 2014 Elsevier Inc. All rights reserved.
polymers used as pharmaceutical excipients and have been used in approved prescription and over-the-counter drug products in the United States and globally for many years. PVAP and PVAP-T are typically used in fully formulated dry powder coating systems manufactured by Colorcon, such as SuretericÒ and CoatericÒ, for rapid reconstitution providing an efficient method of applying enteric film coatings. Extensive toxicological testing of PVAP was conducted by Merck with PVAP in the 1960’s and Colorcon was able to secure reports of these studies. The reports did not describe the characterization, specifications or manufacturing process for the test material used in the studies. The studies were conducted consistent with generally accepted guidelines at that time. The studies were critically evaluated by experts and their findings published (Schoneker et al., 2003). A series of safety studies with PVAP manufactured by Colorcon to NF specifications which included a definitive 90-day sub-chronic toxicity study, a developmental toxicity study and several genotoxicity tests consistent with current Good Laboratory Practice (GLP) regulations and internationally recognized guidelines were conducted to provide additional safety data to support current and new applications for PVAP and PVAP-T. The results of the developmental toxicity study are reported herein. The 90-day sub-chronic toxicity study and the genotoxicity tests were accepted for publication in Food and Chemical Toxicology (DeMerlis et al., 2014).
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Fig. 1. PVAP chemical structure. Depending on the phthalyl content (a) will vary with (b) in mole percent. The acetyl content (c) remains constant depending on the starting material.
The purpose of this developmental toxicity study was to detect adverse effects of polyvinyl acetate phthalate (PVAP) on Crl:CD(SD) presumed-pregnant female rats and on embryo/fetal development consequent to exposure of the dam from implantation to closure of the hard palate. 2. Materials and methods This study was designed to evaluate ICH Harmonised Tripartite Guideline stages C and D of the reproductive process. The requirements of the U.S. Food and Drug Administration (FDA) and the Organisation for Economic Co-operation and Development were also used as the basis for study design (FDA Redbook, 2000; OECD 414, 2001; FDA, 2005; (ICH) S5(R2), 2005). 2.1. Test material Commercial samples of PVAP (NF) CAS No. 34481-48-6 were provided by Colorcon Inc. West Point, PA. The test material was a white to off-white powder and was stored at room temperature with a desiccant. Information documenting or certifying the identity, composition, strength, activity/purity and stability of the test article was provided by Colorcon to the testing facility. Neither the sponsor nor the study director was aware of any potential contaminants likely to have been present in the carrier that would have interfered with the results of this study. 2.2. Animals and husbandry Female rats Crl:CD(SD) were supplied from Charles River Laboratories, Inc., Portage, MI, USA. Male rats were used only for the purposes of breeding and are not considered part of the test system. The Crl:CD(SD) rat was selected as the test system because it is one mammalian species accepted and widely used world-wide for nonclinical studies of developmental toxicity (embryo-fetal toxicity/teratogenicity). This strain has been demonstrated to be sensitive to developmental toxicants and historical data and experience exist at the testing facility used for the study (Christian and Voytek, 1982; Christian, 1984; Lang, 1988). One hundred presumed pregnant Crl:CD(SD) rats with a mean age of 66 days were randomly assigned to four exposure groups (Groups I through IV), 25 rats per group. Mean weight (g ± SD) at study assignment (DG 0) was 231 ± 10.0. Upon arrival, rats were assigned to individual housing on the basis of computer-generated random units. Healthy, mated female rats were assigned to four exposure groups (Groups I through IV), 25 rats per group, using a computer-generated (weight-ordered) randomization procedure based on body weights recorded on DG 0. Male rats were given unique permanent identification numbers upon assignment to the testing facility’s breeder male rat population. Breeder rats were permanently identified using a tail tattoo.
Female rats were assigned temporary numbers at receipt and given unique permanent identification numbers when assigned to the study. Cage tags were marked with the study number, permanent rat number, sex, generation, test article identification, group number and dosage level. During the course of the study, individual rats were examined by the veterinary staff when needed. Medical treatments consisted of application of hydrogen peroxide to scabs. The study rooms were maintained under conditions of positive airflow relative to a hallway and independently supplied with a minimum of ten changes per hour of 100% fresh air that had been passed through 99.97% HEPA filters. Room temperature and humidity were monitored constantly throughout the study. Room temperature was targeted at 64 °F to 79 °F (18 °C to 26 °C); relative humidity was targeted at 30–70%. Rats were individually housed in stainless steel, wire-bottomed cages, except during the cohabitation period. During cohabitation, each pair of male and female rats was housed in the male rat’s cage. All cage sizes and housing conditions were in compliance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources). An automatically controlled 12-h dark:12-h fluorescent light cycle was maintained. Each dark period began at 1900 h (±30 min). Formulated diets of the test article, PVAP, in Certified Rodent DietÒ (meal form) #5002 (PMIÒ Nutrition International, St. Louis, MO, USA) were administered at a constant concentration of the test article in the diet (mg of PVAP/gram of diet). Female rats were given continual access to the formulated diets on days 6 through 20 of presumed gestation (DGs 6 through 20) at concentrations of 0%, 0.75%, 1.5% and 3%. All surviving rats were sacrificed and Caesarean-sectioned on DG 21. The following parameters were evaluated: viability, clinical observations, body weights, feed consumption, necropsy observations, Caesarean-sectioning and litter observations, including gravid uterine weights, fetal body weights and sex, and fetal gross external, soft tissue and skeletal alterations. 2.3. Test diet preparation Diets were prepared once at the testing facility, stored at controlled room temperature (20 °C to 25 °C) and used within the 43-day stability period. A constant concentration of the test article in the diet (mg of PVAP/gram of diet) was administered to the rats. Control group samples were determined to have no detectable level of PVAP. The formulations were within the acceptable limits of ±15% of theoretical concentrations and within the acceptable limits of ±5% RSD for homogeneity. Stability data for test article diet formulations bracketing the range of concentrations in this study were determined under the CRO’s protocol. Rats were provided ad libitum access to Certified Rodent DietÒ #5002 (PMIÒ Nutrition International, St. Louis, MO, USA) and/or prepared diets of Certified Rodent DietÒ #5002 and the test article in individual feeders. Dietary analyses revealed that no contaminants at levels exceeding the maximum concentration limits for certified feed or deviations from expected nutritional requirements were present. Local water that had been processed by passage through a reverse osmosis membrane (R.O. water) was available to the rats ad libitum from an automatic watering access system. Chlorine (sodium hypochlorite) was added to the processed water at a targeted concentration of 0.05 to 1.1 ppm as a bacteriostat. The processed water was analyzed twice annually for possible chemical contamination and monthly for possible bacterial contamination. Chewable NylabonesÒ were supplied to all rats during the course of the study. Environment enrichment is recommended by the Guide for the Care and Use of Laboratory Animals (1996). Analyses for possible contamination were conducted on each lot of NylabonesÒ and documented in the raw data.
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2.4. Experimental design Exposure Group
Concentrationa (%)
Number of rats
I II III IV
0.00 0.75 1.50 3.00
25 25 25 25
a The test article was considered 100% active/pure for the purpose of concentration calculations.
The oral (diet) route was selected for use because it is the proposed route for consumers. Dosages were selected on the basis of a 14-day range-finding toxicity study of PVAP administered in the diet to male and female Crl:CD SD rats. In that study, administration of PVAP in the diet for a minimum of 14 days was well tolerated in rats up to the highest level tested, 5% equivalent to 4.16 g/kg/day. Other concentrations tested were 0%, 1%, 2%, 3% PVAP. Mean test article consumption was 0.68, 1.40, 2.11, and 3.56 g/kg/day for treated males, and 0.80, 1.58, 2.44, and 4.16 g/kg/day for treated females. There was no test article-related mortality observed in this study. No test article-related clinical signs were noted. There were no toxicologically meaningful differences in body weight, body weight changes, food consumption, hematology parameters, coagulation parameters, clinical chemistry parameters, urinalysis parameters (macroscopic and microscopic), or absolute and relative organ weights in test article-treated males or females compared to controls. No test article-related gross findings were observed at scheduled sacrifice. Based on these results, the no-observedadverse-effect level (NOAEL) was determined to be 5% equivalent to 3.56 g/kg/day for males and 4.16 g/kg/day for females. Female rats were given continual access to the formulated diets on DGs 6 through 20. A constant concentration of the test article in the diet (mg of PVAP/gram of diet) was administered to the rats. The mg/kg/day dosages consumed were calculated based on the body weights and feed consumption values of the rats and presented for periods corresponding to body weight and feed consumption observations. 2.5. In-life observations After acclimation (5 days), 120 virgin female rats were cohabitated with 120 breeder male rats, one male rat per female rat. The cohabitation period consisted of a maximum of five days. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be DG 0 and assigned to individual housing. Mating was evaluated daily during the cohabitation period and confirmed by observation of spermatozoa in a smear of the vaginal contents and/or a copulatory plug observed in situ. Rats were observed for viability at least twice each day of the study and for clinical observations and general appearance weekly during the acclimation period and on DG 0. The rats were also examined for clinical observations, abortions, premature deliveries and deaths once daily during the exposure and postexposure periods. Body weights were recorded weekly during the acclimation period, on DG 0 and daily during the exposure and postexposure periods. Feed consumption values were recorded on DG 0 and daily during the exposure and postexposure periods. 2.6. Gross necropsy To minimize bias, Caesarean-sectioning, necropsy and subsequent fetal observations were conducted without knowledge of exposure group. Rats were euthanized by carbon dioxide asphyxiation. Live fetuses were euthanized by an intraperitoneal injection
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of sodium pentobarbital. Gross lesions were retained in neutral buffered 10% formalin for possible future evaluation. Unless specifically cited below, all other tissues were discarded. Representative photographs of maternal gross lesions and fetal gross, soft tissue and skeletal alterations are available in the raw data. All surviving female rats were euthanized on DG 21, Caesareansectioned and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. The gravid uterus was excised and weighed. One rat was apparently non-pregnant and the uterus was stained with 10% ammonium sulfide (Salewski, 1964) to confirm the absence of implantation sites. The uterus and ovaries of the apparently non-pregnant rat were retained in neutral buffered 10% formalin and were discarded when authorized by the study director. The number and distribution of corpora lutea were recorded. The uterus of each rat was excised and examined for evidence of pregnancy, number and distribution of implantation sites, early and late resorptions and live and dead fetuses. An early resorption was defined as one in which organogenesis was not grossly evident. A late resorption was defined as one in which the occurrence of organogenesis was grossly evident. A live fetus was defined as a term fetus that responded to stimuli. Non-responding term fetuses were considered to be dead. Dead fetuses and late resorptions were differentiated by the degree of autolysis present; marked to extreme autolysis indicated that the fetus was a late resorption. Placentae were examined for size, color and shape. Each fetus was removed from the uterus, placed in an individual container and individually identified with a tag noting the study number, litter number, uterine distribution and fixative. Each fetus was subsequently weighed and examined for sex and gross lesions. Approximately one-half of the fetuses in each litter were examined for soft tissue alterations, using a variation of the microdissection technique of Staples (Staples, 1974; Wilson, 1965). These fetuses were then fixed in Bouin’s solution and the heads were subsequently examined by free-hand sectioning; head sections were stored in alcohol. The decapitated carcasses were discarded. The remaining fetuses (approximately one-half of the fetuses in each litter) were eviscerated, cleared, stained with alizarin red S (Staples and Schnell, 1964) and examined for skeletal alterations. The fetuses were initially fixed in alcohol. Skeletal preparations were retained in glycerin with thymol added as a preservative. The rat that was sacrificed before scheduled termination due to adverse clinical observations was examined for the cause of condition on the day the observation was made. The rat was examined for gross lesions. The heart, lungs, liver, kidneys, stomach and spleen were retained in neutral buffered 10% formalin for possible histological evaluation. Pregnancy status and uterine contents were recorded. Conceptuses in utero were examined to the extent possible, using the same methods described for term fetuses. 2.7. Statistical analyses Data generated during the course of this study were recorded either by hand or using the Argus Automated Data Collection and Management System and the Vivarium Temperature and Relative Humidity Monitoring System. All data were tabulated, summarized and/or statistically analyzed. Averages and percentages were calculated. Litter values were used where appropriate. Clinical observations and other proportional data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution (Snedecor and Cochran, 1967a). Continuous data (e.g., body weights, body weight changes, feed consumption values, organ weights and litter averages for percent male fetuses, percent resorbed conceptuses, fetal body weights and fetal anomaly data) were analyzed using Bartlett’s Test of Homogeneity of Variances (Sokal and Rohlf, 1969a) and the Analysis of
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Variance (Snedecor and Cochran, 1967b), when appropriate [i.e., Bartlett’s Test was not significant (p > 0.001)]. If the Analysis of Variance was significant (p 6 0.05), Dunnett’s Test (Dunnett, 1955) was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett’s Test was significant (p 6 0.001)], the Kruskal–Wallis Test (Sokal and Rohlf, 1969b) was used, when less than or equal to 75% ties were present. In cases where the Kruskal–Wallis Test was statistically significant (p 6 0.05), Dunn’s Method of Multiple Comparisons (Dunn, 1964) was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher’s Exact Test (Siegel, 1956) was used to analyze the data. Count data were evaluated using the procedures described above for the Kruskal–Wallis Test (Sokal and Rohlf, 1969b). 3. Results There was no test article-related mortality. One female rat in the 0% exposure group was euthanized due to a broken hindlimb. All other rats survived to scheduled sacrifice. There were no test article related clinical or necropsy observations. Body weights, body weight gains, absolute and relative feed consumption and gravid uterine weights were unaffected by concentrations of PVAP in the diet as high as 3% (Figs. 2–4, Tables 1–3). The slight increase in feed consumption in the treated rats consuming PVAP may be due to the presence of the non-nutritive PVAP in the diet. No Caesarean-sectioning or litter parameters were affected by concentrations of PVAP in the diet as high as 3%. No gross external, soft tissue or skeletal fetal alterations (malformations or variations) were caused by concentrations of PVAP in the diet as high as 3%. Fetal ossification site averages were comparable among the four dosage groups and did not significantly differ.
450
Weight (g)
400 350 300
0.00% 0.75% 1.50% 3.00%
250 200
0
3
6
9 12 Days of gestation
15
18
Absolute consumaption (g)
Fig. 2. Maternal body weight.
35 30 25 20 0.00% 0.75% 1.50% 3.00%
15 10 5 0
0-6
6-9
9-12 12-15 Days of gestation
15-18
18-21
Fig. 3. Summary of maternal absolute feed consumption values.
100
Relative consumaption (g)
328
80 60 0.00% 0.75% 1.50% 3.00%
40 20 0
0-6
6-9
9-12
12-15
15-18
18-21
Days of gestation Fig. 4. Summary of maternal relative feed consumption values.
Consumed dosages averaged 567.0, 1139.1 and 2324.6 mg/kg/ day in Groups II, III and IV, respectively, for the entire dosage period (gestation days 6–20). 3.1. Mortality There was no test article-related mortality. One female rat in the 0% exposure group was euthanized due to a broken hindlimb. Observations in this rat are described in the following paragraph. All other rats survived to scheduled termination of the study. One rat in the 0% exposure group was euthanized due to adverse clinical observations on day 15 of gestation (DG 15). Adverse clinical observations occurred only on DG 15 and consisted of impaired righting reflex, decreased motor activity, chromodacryorrhea, chromorhinorrhea, lacrimation, limited use of both hindlimbs, limited gripping reflex in left hindlimb, moderate dehydration, cold to touch, tachypnea, urine-stained abdominal fur and scant feces. This rat lost 9.2% of the DG 13 body weight by DG 15. Feed consumption was reduced from DGs 13 to 15. Because these reduced values reflected this injury, body weights and feed consumption values on DG 14 and 15 were excluded from group averages and statistical evaluation. Necropsy revealed a fracture in the left fibula; all other tissues appeared normal. The litter consisted of eight embryos and six early resorptions. There was no obvious cause of the broken hindlimb; it is likely the rat got the limb caught in the cage and/or feed jar and injured itself trying to free it. 3.2. Clinical and necropsy observations
21
Adverse clinical observations were considered unrelated to PVAP because the incidences were not dosage dependent and/or the observations occurred in only one rat in a group. These clinical observations included sparse hair coat, chromorhinorrhea, scab on the nose, localized alopecia, bent tail, impaired righting reflex, decreased motor activity, chromodacryorrhea, lacrimation, limited use of hindlimb, limited gripping reflex of hindlimb, moderate dehydration, cold to touch, tachypnea, urine-stained abdominal fur and scant feces. Abnormal necropsy observations were considered unrelated to PVAP because the incidences were not dosage dependent and/or the observations occurred in only one rat in a group. The necropsy observations included large lymph nodes, numerous red areas on the thymus, slight dilation of the renal pelvis, large spleen and fractured fibula. Statistically significant increases (p 6 0.05 or p 6 0.01) in absolute and relative feed consumption in the 1.5% and 3% exposure groups on DGs 12 to 15, in relative feed consumption in the 1.5% and 3% exposure groups on DGs 9 to 12 and 6 to 21, and in relative feed consumption in the 3% exposure group on DGs 0 to 21 were
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C.C. DeMerlis et al. / Regulatory Toxicology and Pharmacology 70 (2014) 325–332 Table 1 Summary of maternal body weights and gravid uterine weights (tested rats/group: 25. pregnant rats/group: 25). Exposure group Concentrationa (%) Days of gestation
I 0.00 Maternal body weight (G)
II 0.75
III 1.50
IV 3.00
0 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 21Cd
231.5 ± 10.1 264.2 ± 11.5 269.0 ± 11.9 273.8 ± 11.7 279.7 ± 12.7 286.4 ± 12.8 292.6 ± 12.8 297.8 ± 12.5 304.4 ± 12.4 312.0 ± 12.5b,c 321.3 ± 12.6b,c 331.3 ± 12.2b,e 345.7 ± 12.8b,e 362.3 ± 12.8b,e 379.0 ± 13.4b,e 397.1 ± 13.7b,e 423.8 ± 15.7b,e 318.7 ± 13.5b,e
231.0 ± 10.2 263.8 ± 12.3 269.1 ± 13.5 273.3 ± 13.7 281.2 ± 14.2 287.9 ± 14.8 294.5 ± 15.7 301.1 ± 16.2 307.2 ± 16.0 313.4 ± 17.4 322.6 ± 18.3 333.1 ± 18.1 346.4 ± 19.9 363.0 ± 21.3 378.6 ± 22.2 395.9 ± 23.8 419.6 ± 23.1 317.0 ± 19.1
231.6 ± 9.5 265.4 ± 12.5 270.6 ± 13.2 276.4 ± 13.7 282.9 ± 14.1 289.1 ± 14.2 297.2 ± 15.0 302.6 ± 15.9 308.8 ± 16.2 314.9 ± 16.6 324.6 ± 17.4 335.6 ± 17.2 349.4 ± 18.7 365.6 ± 18.6 381.4 ± 19.8 399.1 ± 19.7 425.0 ± 21.8 320.4 ± 20.5
230.6 ± 11.0 263.3 ± 15.5 269.5 ± 15.4 274.9 ± 16.4 279.2 ± 17.2 285.6 ± 17.2 294.1 ± 18.2 300.0 ± 18.2 306.3 ± 19.0 313.2 ± 18.4 321.9 ± 19.7 332.4 ± 20.7 345.8 ± 20.9 362.0 ± 21.7 376.8 ± 23.2 392.4 ± 23.7 417.0 ± 24.8 317.1 ± 21.8
102.7 ± 13.3
104.6 ± 10.0
99.9 ± 18.2
Gravid uterine weight (G) 105.1 ± 9.9b,e a b c d e
Rats were given continual access to the formulated diets on days 6 through 20 of gestation. Average number of tested/pregnant rats: 24. Excludes values for dam 17553 that had reduced body weight values after 13 days of gestation reflecting an injury. Corrected maternal body weight (day 21 of gestation body weight minus the gravid uterine weight). Excludes values for dam 17553 that was sacrificed due to adverse clinical observations.
Table 2 Summary of maternal absolute feed consumption values (tested rats/group: 25; pregnant rats/group: 25 except 24 rats in group IV).
Table 3 Summary of maternal relative feed consumption values (tested rats/group: 25; pregnant rats/group: 25 except 24 rats in group IV).
Exposure group Concentrationa (%) Days of gestation
I II III 0.00 0.75 1.50 Maternal feed consumption (G/Day)
IV 3.00
Exposure group Concentrationa (%) Days of gestation
I II III 0.00 0.75 1.50 Maternal feed consumption (G/KG/Day)
IV 3.00
0–6 6–9 9–12 12–15 15–18 18–21 6–21 0–21
18.6 ± 1.5 20.9 ± 2.0 22.1 ± 1.9 22.6 ± 1.6b,c 24.9 ± 1.4b,d 24.8 ± 1.7b,d 23.1 ± 1.2b,d 21.8 ± 1.2b,d
18.4 ± 2.0 22.1 ± 2.7 23.7 ± 2.5 24.6 ± 2.8** 26.6 ± 3.5 24.9 ± 3.6 24.4 ± 2.4 22.6 ± 2.1
0–6 6–9 9–12 12–15 15–18 18–21 6–21 0–21
75.2 ± 4.9 76.8 ± 5.3 76.5 ± 4.9 73.3 ± 4.5b,c 73.2 ± 4.4b,d 63.5 ± 4.1b,d 71.7 ± 2.9b,d 68.9 ± 2.7b,d
74.5 ± 5.5 81.2 ± 7.3 81.6 ± 6.2** 79.0 ± 6.5** 77.9 ± 8.2 64.2 ± 8.0 75.8 ± 5.0** 71.7 ± 4.2**
18.7 ± 1.6 21.8 ± 2.1 23.0 ± 2.3 23.7 ± 2.2 25.7 ± 2.2 24.5 ± 2.4 23.7 ± 1.9 22.3 ± 1.7
18.7 ± 1.6 22.1 ± 1.5 23.4 ± 1.6 24.0 ± 1.8* 25.9 ± 2.4 25.2 ± 2.3b,e 24.0 ± 1.6b,e 22.4 ± 1.4b,e
75.4 ± 4.6 80.1 ± 6.1 79.1 ± 5.7 76.1 ± 5.0 75.4 ± 5.2 63.1 ± 5.7 73.7 ± 4.4 70.4 ± 3.6
75.3 ± 5.4 80.7 ± 4.9 79.7 ± 3.7* 76.8 ± 4.4* 75.4 ± 5.1 64.2 ± 4.5b,e 74.2 ± 3.2b,e,* 70.6 ± 2.5b,e
a Rats were given continual access to the formulated diets on days 6 through 20 of gestation. b Average number of tested/pregnant rats: 24. c Excludes values for dam 17553 that had reduced feed consumption values after day 13 of gestation reflecting an injury. d Excludes values for dam 17553 that was sacrificed due to adverse clinical observations. e Excludes values that were associated with spillage. * Significantly different from the Group I value (p 6 0.05). ** Significantly different from the Group I value (p 6 0.01).
a Rats were given continual access to the formulated diets on days 6 through 20 of gestation. b Average number of tested/pregnant rats: 24. c Excludes values for dam 17553 that had reduced feed consumption values after day 13 of gestation reflecting an injury. d Excludes values for dam 17553 that was sacrificed due to adverse clinical observations. e Excludes values that were associated with spillage. * Significantly different from the Group I value (p 6 0.05). ** Significantly different from the Group I value (p 6 0.01).
not considered to be toxicologically significant because decreased feed consumption, rather than increased, is the expected effect of a toxicant. There were 25 (100%), 25 (100%), 25 (100%) and 24 (96%) pregnant rats in Groups I through IV, respectively. As a result of the sacrifice of the control group dam with a broken hindlimb, Caesarean-sectioning observations were based on 24, 25, 25 and 24 pregnant rats in the four respective groups. There were no PVAP-related adverse effects on Caesareansectioning or litter parameters affected by concentrations of PVAP in the diet as high as 3% (Tables 4 and 5). The litter means for corpora lutea, implantations, preimplantation loss, litter sizes, live and dead fetuses, early and late resorptions, postimplantation loss, fetal body weights, percent dead or resorbed conceptuses, and percent
live male fetuses were comparable among the four dosage groups and did not differ significantly. There was one dead fetus in the 3% exposure group and one dam in the 3% exposure group that had a litter consisting of all late resorptions. These observations were not considered to be test article related because they were single events and not dose-dependent. One control group dam had three enlarged placentae (out of a total of 14). All other placentae appeared normal. Fetal alterations were defined as malformations (irreversible changes that occur at low incidences in this species and strain) or variations (common findings in this species and strain and reversible delays or accelerations in development). Litter averages were calculated for specific fetal ossification sites as part of the evaluation of the degree of fetal ossification.
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Table 4 Summary of Caesarean-sectioning observations. Exposure group Concentrationa (%)
I 0.00
II 0.75
III 1.50
IV 3.00
Rats tested Pregnant rats (number/%) Unscheduled sacrifice (number/%) Rats pregnant and caesarean-sectioned on day 21 of gestation
25 25/100 1/4 24
25 25/100 0/0 25
25 25/100 0/0 25
25 24/96 0/0 24
Corpora lutea
15.4 ± 1.6
14.9 ± 1.8
16.4 ± 2.8
14.7 ± 2.0
Implantations Preimplantation loss (%)
14.6 ± 1.4 4.9 ± 5.8
14.2 ± 1.9 4.4 ± 6.3
15.0 ± 1.4 6.7 ± 12.8
14.0 ± 1.6 4.1 ± 6.0
Litter sizes Live fetuses
14.1 ± 1.4 339 14.1 ± 1.4 0 0.0 ± 0.0
13.7 ± 2.1 343 13.7 ± 2.1 0 0.0 ± 0.0
14.1 ± 1.6 353 14.1 ± 1.6 0 0.0 ± 0.0
13.1 ± 3.2 314 13.1 ± 3.2 1 0.0 ± 0.2
Postimplantation loss (%)
0.4 ± 0.6 11 0.4 ± 0.6 0 0.0 ± 0.0 3.0 ± 4.4
0.5 ± 1.0 13 0.5 ± 1.0 0 0.0 ± 0.0 3.6 ± 6.8
0.9 ± 1.2 23 0.9 ± 1.2 0 0.0 ± 0.0 6.0 ± 7.5
0.9 ± 2.6 7 0.3 ± 0.6 15 0.6 ± 2.6 7.1 ± 20.4
Dams with any resorptions (number/%) Dams with all conceptuses dead or resorbed (number/%) Dams with viable fetuses (number/%) Placentae appeared normalb (number/%)
9/37.5 0/0.0 24/100.0 23/95.8
8/32.0 0/0.0 25/100.0 25/100.0
13/52.0 0/0.0 25/100.0 25/100.0
8/33.3 1/4.2 23/95.8 23/100.0
Dead fetuses Resorptions Early resorptions Late resorptions
% preimplantation loss = [(number of corpora lutea number of implantations)/number of corpora lutea] 100. % postimplantation loss = [(number of implantations number of live fetuses)/number of implantations] 100. a Rats were given continual access to the formulated diets on days 6 through 20 of gestation. b Excludes values for dams with all late resorptions.
Table 5 Summary of litter observations (Caesarean-delivered fetuses). Exposure group Concentrationa (%)
I 0.00
II 0.75
III 1.50
IV 3.00
Litters with one or more live fetuses
24
25
25
23
Implantations Live fetuses
14.6 ± 1.4 339 14.1 ± 1.4 52.6 ± 10.5 5.46 ± 0.23 5.58 ± 0.22 5.31 ± 0.25 3.00 ± 4.40
14.2 ± 1.9 343 13.7 ± 2.1 54.2 ± 14.2 5.53 ± 0.28 5.67 ± 0.30 5.37 ± 0.30 3.60 ± 6.80
15.0 ± 1.4 353 14.1 ± 1.6 49.8 ± 12.4 5.46 ± 0.25 5.62 ± 0.24 5.30 ± 0.25 6.00 ± 7.50
14.1 ± 1.6 314 13.6 ± 1.7 49.0 ± 14.3 5.50 ± 0.33 5.66 ± 0.36 5.35 ± 0.32 3.10 ± 5.30
Live male fetuses/litter (%) Live fetal body weights/litter (g) Males fetuses (g) Female fetuses (g) Dead or resorbed conceptuses/litter (%) a
Rats were given continual access to the formulated diets on days 6 through 20 of gestation.
Table 6 Summary of fetal observations. Exposure group Concentration (%)
I 0
II 0.75
III 1.5
IV 3
Litters with fetuses with alterations Numbers of fetuses with alterations Percent of fetuses with alterations Bifid centrum in thoracic vertebra (fetuses, litters)
6
5
6
3
7
7
6
3
2
2.2
1.7
0.9
2.2
1.1
4.4
1.1
Innominate artery absent
Innominate artery absent
Constricted tail; no anal opening
Moderate dilation of ureters
Innominate artery absent Short ribs
Interfrontal ossification site Cervical rib at 7th cervical vertebra
Folded retina Arches of 6th cervical vertebra had the appearance of the 7th
Incomplete ossified cervical arch Cervical rib at 7th cervical vertebra
Additional Observations
2 littermates with wavy ribs; one with incomplete ossified rib
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Fetal evaluations were based on 339, 343, 353 and 314 live, DG 21 caesarean-delivered fetuses in 24, 25, 25 and 23 litters in the 0, 0.75%, 1.5% and 3% exposure groups, respectively. Each of these fetuses was examined for gross external alterations. Of these respective fetuses, 161, 164, 170 and 150 fetuses were examined for soft tissue alterations, and 178, 179, 183 and 164 fetuses were examined for skeletal alterations and fetal ossification site averages. It was also possible to examine one dead fetus in the 3% exposure group. This specimen had a medially rotated left hindlimb and a laterally rotated right hindlimb at external examination and all tissues appeared normal at soft tissue examination. Values for this specimen were excluded from summarization and statistical analyses. There were no PVAP-related gross external, soft tissue or skeletal fetal alterations (malformations or variations). Mean fetal ossification sites averages were comparable among the four dosage groups and did not differ significantly. There were no statistically significant or biologically important differences among the four dosage groups in the average numbers of ossification sites per fetus for the hyoid, vertebrae (cervical, thoracic, lumbar, sacral and caudal), ribs, sternum (manubrium, sternal centers and xiphoid), forelimbs (carpals, metacarpals and phalanges) or hindlimbs (tarsals, metatarsals and phalanges). All values were within the ranges observed historically at the testing facility. Fetal observations are included in Table 6.
4. Discussion and conclusion PVAP, polyvinyl acetate phthalate, a phthalate enteric polymer, is a component of enteric coatings used in delayed release tablet coatings. PVAP is not a phthalate as defined by regulatory agencies such as the FDA and EPA. These agencies have defined phthalates as diesters of low molecular weight alcohols and orthophthalic acid (‘‘phthalic acid’’). ‘‘By contrast, phthalate enteric polymers are monoesters of orthophthalic acid and the alcohol is present in the form of a polymer backbone. These differences in structure translate to differences in chemical properties and biological activities.’’ ‘‘The common term in the nomenclature of these high molecular enteric polymers is the word ‘‘phthalate’’ because these enteric polymers have been modified by esterification with orthophthalic acid groups. The enteric polymers are large molecules with a typical number average (Mn) molecular weight in the range of 60,000– 130,000 Da.’’ By contrast, ‘‘esters of orthophthalic acid with low molecular weight alcohols, such as dibutyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) have been developed and used commercially as plasticizers. DBP and DEHP are small molecules with molecular weight of only 278 and 390, respectively’’ (Kossor et al., 2014). The large molecular weight phthalate enteric polymers are not bioavailable following oral administration (gavage, dietary). There are no data to support the metabolism (biotransformation) of these phthalate enteric polymers to bioactive metabolites. Data presented in published in vivo and in vitro studies with Cellulose Acetate Phthalate (CAP), CAP Aqueous Dispersion, Hypromellose Phthalate (HPMCP), and PVAP demonstrate the lack of biological activity of these phthalate enteric polymers. The inappropriate use of the term ‘‘phthalates’’ by some scientists and the media has created unwarranted safety concerns. It is noted, however, that when the FDA recommended to the pharmaceutical industry that it should avoid the use of phthalates, it referred to only two specific phthalates, DBP and DEHP. The weight of the available published evidence on phthalate enteric polymers supports the safety of their use in pharmaceutical preparations. The study reported herein adds further evidence of the biological inertness of these polymers. The toxicity of phthalate enteric
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polymers will not be compared to the toxicity of the phthalates used as plasticizers (e.g. DBP and DEHP) since these phthalates are neither chemically nor functionally equivalent to phthalate enteric polymers and such discussion would only further confuse the scientific literature and the public media. The purpose of this study, a classical developmental study, was to assess the potential developmental toxicity of PVAP in Crl:CD(SD) presumed-pregnant female rats (from implantation to closure of the hard palate). There were no deaths and no evidence of consistent, biologically and/or statistically significant, dosedependent adverse biological effects on any of the parameters evaluated in the dams or fetuses. The maternal and developmental no-observable-adverse-effect level (NOAEL) of PVAP is the highest concentration tested, i.e., 3.0% (equivalent to 2324.6 mg PVAP/kg/day). Conflict of interest C.C. DeMerlis and D.R. Schoneker are employees of Colorcon, Inc. Colorcon retained J.F. Borzelleca as a consultant and compensated him for his services. Acknowledgment The authors thank Hua Deng of Colorcon, Inc. for assistance in the preparation of the manuscript. References Christian, M.S., 1984. Reproductive toxicity and teratology evaluations of naltrexone. J. Clin. Psychiatry 45 (9 Sec 2), 7–10. Christian, M.S., Voytek, P.E., 1982. In Vivo Reproductive and Mutagenicity Tests. A Guide to General Toxicology. S. Karger, Basel (CH), pp. 295–325. DeMerlis, C.C., Schoneker, D.R., Borzelleca, J.F., 2014. Safety of PVAP and PVAP-T including a 90-day dietary toxicity study in rats and genotoxicity tests with polyvinyl acetate phthalate (PVAP). Food Chem. Toxicol. 70, 231–240. Dunn, O.J., 1964. Multiple comparisons using rank sums. Technometrics 6 (3), 241– 252. Dunnett, C.W., 1955. A multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 50, 1096–1121. Guidance for Industry: Nonclinical Studies for the Safety Evaluation of Pharmaceutical Excipients. U.S. Dept of Health and Human Services Food and Drug Administration (CDER) (CBER), Rockville (MD), 2005. Guideline for Industry: Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility (ICH) S5(R2), last update November, 2005. U.S. Dept of Health and Human Services Food and Drug Administration, Rockville (MD). Institute of Laboratory Animal Resources Commission on Life Sciences and the National Research Council, 1996. Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington (DC). Kossor, D., DeMerlis, C., Obara, S., Hernandez, A., Moreton, C., 2014. Eye on Excipients: Misunderstandings about Excipients that Contain ‘‘Phthalate’’ in Their Name. Tablets & Capsules. pp. 39–45. Lang, P.L., 1988. Embryo and Fetal Developmental Toxicity (Teratology) Control Data in the Charles River Crl:CDÒBR Rat. (Database provided by Argus Research Laboratories, Inc.). Charles River Laboratories, Inc, Wilmington (MA). OECD guidelines for the testing of chemicals. Prenatal developmental toxicity study, No. 414 section 4 Health Effects (Pink pages); last updated January 22, 2001. Organisation for Economic Co-operation and Development. Redbook, 2000. Toxicological Principles for the Safety Assessment of Food Ingredients: IV.C.9.b. Guidelines for Developmental Toxicity Studies; July, 2000. U.S. Dept of Health and Human Services Food and Drug Administration (CFSAN) (OFAS), Springfield (VA). Salewski, E.,1964. Färbemethode zum makroskopischen nachweis von implantations stellen am uterus der ratte. G [Staining method for macroscopic demonstration of implantation sites in the rat uterus]. Arch. Pathol. Exp. Pharmacol. 247, 367. Schoneker, D.R., DeMerlis, C.C., Borzelleca, J.F., 2003. Evaluation of the toxicity of polyvinyl acetate in experimental animals. Food Chem. Toxicol. 41, 405. Siegel, S., 1956. The Fisher’s Exact Probability Test. Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill Co, New York (NY), pp. 96–105. Snedecor, G.W., Cochran, W.G., 1967a. Variance Test for Homogeneity of the Binomial Distribution. Statistical methods, sixth ed. Iowa State University Press, Ames, pp. 240–241. Snedecor, G.W., Cochran, W.G., 1967b. Analysis of Variance. Statistical methods, sixth ed. Iowa State University Press, Ames, pp. 258–298.
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